RESEARCH ARTICLEOFFICIAL JOURNAL

www.hgvs.org

Assessment of the Potential Pathogenicity of MissenseMutations Identified in the GTPase-Activating Protein(GAP)-Related Domain of the Neurofibromatosis Type-1(NF1) Gene

Laura Thomas, Mark Richards, Matthew Mort, Elaine Dunlop, David N. Cooper, and Meena Upadhyaya∗

Institute of Medical Genetics, Cardiff University, Cardiff, UK

Communicated by Jacques S. BeckmannReceived 10 May 2012; accepted revised manuscript 28 June 2012.Published online 16 July 2012 in Wiley Online Library (www.wiley.com/humanmutation).DOI: 10.1002/humu.22162

ABSTRACT: Neurofibromatosis type-1 (NF1) is causedby constitutional mutations of the NF1 tumor-suppressorgene. Although ∼85% of inherited NF1 microlesionsconstitute truncating mutations, the remaining ∼15%are missense mutations whose pathological relevance isoften unclear. The GTPase-activating protein-relateddomain (GRD) of the NF1-encoded protein, neurofi-bromin, serves to define its major function as a negativeregulator of the Ras-MAPK (mitogen-activated pro-tein kinase) signaling pathway. We have established afunctional assay to assess the potential pathogenicityof 15 constitutional nonsynonymous NF1 missensemutations (11 novel and 4 previously reported butnot functionally characterized) identified in the NF1-GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R,p.I1307V, p.T1324N, p.E1327G, p.Q1336R,p.E1356G, p.R1391G, p.V1398D, p.K1409E,p.P1412R, p.K1436Q, p.S1463F). Individual muta-tions were introduced into an NF1-GRD expressionvector and activated Ras was assayed by an enzyme-linkedimmunosorbent assay (ELISA). Ten NF1-GRD variantswere deemed to be potentially pathogenic by virtue ofsignificantly elevated levels of activated GTP-bound Rasin comparison to wild-type NF1 protein. The remainingfive NF1-GRD variants were deemed less likely to be ofpathological significance as they exhibited similar levelsof activated Ras to the wild-type protein. These conclu-sions received broad support from both bioinformaticanalysis and molecular modeling and serve to improve ourunderstanding of NF1-GRD structure and function.Hum Mutat 00:1–10, 2012. C© 2012 Wiley Periodicals, Inc.

KEY WORDS: NF1; GAP-related domain; bioinformaticanalysis; molecular modeling

Additional Supporting Information may be found in the online version of this article.∗Correspondence to: Meena Upadhyaya, Institute of Medical Genetics, School of

Medicine, Cardiff University, Cardiff, CF14 4XN, UK. E-mail: upadhyaya@cardiff.ac.uk

Contract grant sponsor: Cardiff University Cancer IRG.

IntroductionThe human neurofibromatosis type-1 gene, (NF1; MIM# 162200)

a tumor suppressor located on chromosome 17q11.2, comprises 61exons and encodes neurofibromin, a 2818 amino acid protein whichis expressed at a high level in the brain and central nervous system.Neurofibromin is a highly conserved Ras-GTPase-activating protein(Ras-GAP) that functions as a negative regulator of the Ras-MAPK(mitogen-activated protein kinase) signaling pathway [Gutmannet al., 1993; Upadhyaya, 2010]. Neurofibromin downregulates thebiological activity of normal (but not oncogenic) Ras proteins by act-ing as a GTPase-activating protein (GAP) [Ballester et al., 1990; Xuet al., 1990; Martin et al., 1990]. The 413 amino acid (NP_000258.1;p.1125–1537) GAP-related domain (GRD), encoded by exons 20 to27a of the NF1 gene (NM_000267), constitutes the major functionaldomain of neurofibromin.

Neurofibromin displays extensive sequence homology to twomammalian RAS-GTPases (including p21ras) as well as to Ira1p andIra2p, two inhibitory regulators of the Ras-cAMP pathway identifiedin Saccharomyces cerevisiae [Andersen et al., 1993]. The downreg-ulation of active Ras by GAP is a crucial step in the regulation ofthe Ras-MAPK signaling pathway and is disrupted in a significantproportion of different cancers [Adjei, 2001]. Neurofibromin bindsto Ras proteins through its GRD; binding promotes GTP hydrolysisby inducing the intrinsic GTPase activity of Ras thereby convert-ing Ras from its active GTP-bound form to an inactive GDP-boundform. This serves to downregulate Ras-MAPK signaling, with down-stream consequences for a variety of cellular processes including cel-lular proliferation and DNA synthesis [Arun and Gutmann, 2004;Ballester et al., 1990; Cichowski and Jacks, 2001; DeClue et al., 1991;Gottfried et al., 2006; Martin et al, 1990; Xu et al., 1990].

The Ras-GAP activity of neurofibromin has been investigatedboth structurally and biochemically. A number of regions withinNF1-GAP interact with Ras; these include the arginine-finger loop,the Phe-Leu-Arg (FLR) region and the α7/variable loop region [Ah-madian et al., 2003]. These regions undergo conformational changesbetween the active and inactive states and are thought to interactwith Ras by binding to the switch I region through the α7/variableloop, and by presenting an arginine residue (R1276 in neurofi-bromin) to the catalytic region of Ras [Ahmadian et al., 2003]. Theunderlying mechanism of GTPase stimulation involves the stabi-lization of (1) amino acid residues in the switch I and switch IIregions of Ras and (2) the transition state of GTP hydrolysis by neu-tralization of the developing negative charge on the GTP phosphateoxygen atoms during phosphoryl transfer. This occurs through theinsertion of the catalytic arginine (R1276) into the active site of Ras

C© 2012 WILEY PERIODICALS, INC.

[Morgan et al., 2007; Resat et al., 2001; Scheffzek et al., 1997, 1998b;Welti et al., 2008].

The NF1 gene has one of the highest mutation rates reported forany locus associated with an inherited human disorder [Huson et al.,1989]. For this reason, the germline mutational spectrum is quitewell defined, with at least 1,348 different NF1 gene lesions char-acterized to date [see Human Gene Mutation Database (HGMD);http://www.hgmd.org]. Loss of function of the NF1 gene throughmutation leads to increased activity of the Ras-MAPK pathway (as aconsequence of the loss of negative regulation), which serves to pro-mote tumorigenesis. At least 150 of the known NF1 gene lesions arelocated within the GRD of neurofibromin, including 35 missensemutations. The functional characterization of these naturally occur-ring missense mutations has yielded new insights into the structureand function of the GRD [Ahmadian et al., 2003; Klose et al., 1998;Li et al., 1992; Morcos et al., 1996; Poullet et al., 1994; Scheffzeket al., 1996, 1997; Skinner et al., 1991; Upadhyaya et al., 1997].Thus, for example, Li et al. (1992) analyzed the K1423E mutationby site-directed mutagenesis, in vitro expression and subsequent as-say measurement of phosphate release. The GAP activity displayedby the mutant NF1-GRD was 200–400-fold lower than that of thewild type although the binding affinity was unaffected. In principle,such biochemical studies of key residues within the GRD have thepotential to provide new information on the role of these residuesin Ras–Ras-GAP interactions. However, to date, only four natu-rally occurring NF1-GRD mutants (K1423E, R1391K, R1391S, andR1276P) have been functionally analyzed [Ahmadian et al., 2003,Klose et al., 1998; Li et al., 1992; Morcos et al., 1996; Poullet et al.,1994; Upadhyaya et al., 1997].

Another tool to aid the structural and functional characteriza-tion of NF1-GRD missense mutations is molecular modeling. Thethree-dimensional structure of the GRD presents an entirely helicalprotein with a groove on the surface [Scheffzek et al., 1996; 1998a,b].This groove, which contains evolutionarily highly conserved aminoacid residues, serves to accommodate Ras within the interaction ofRas and Ras-GAP [Morcos et al., 1996; Scheffzek et al., 1996, 1997].Although a basic knowledge of Ras–Ras-GAP interactions has beenacquired from such studies, several issues are worthy of further in-vestigation, for example, the identity of the amino acid residuesin neurofibromin that are involved in mediating NF1-GAP’s affin-ity for Ras, catalysis by the arginine finger, and stabilization of theswitch regions in Ras. Here, we report (1) the identification of 11novel missense mutations, putatively responsible for causing neu-rofibromatosis type-1 (NF1), that are located within the GRD ofneurofibromin and (2) the combined functional and bioinformaticanalysis of a total of 17 GRD missense variants. Taken together,this study constitutes the most comprehensive functional analysisof NF1-GRD mutations performed to date.

Materials and Methods

Plasmid Preparation

The basic plasmid, 19993 pGBT9-NF1-GRD, containing theGTPase-activating protein (GAP)-related domain (GRD) of the NF1gene [NM_000267] (residues 3538–4512), was obtained from Ad-dgene (Cambridge, MA) [Morcos et al., 1996]. Gateway Technologywith Clonase II (Invitrogen) was then used, following the manufac-turers’ instructions, to clone the plasmid DNA into pcDNA3.2/V5-DEST (Invitrogen, Paisley). The following primers were used togenerate PCR products suitable for use in the Gateway BP re-combination reaction: Forward: 5′-GGGACAAGTTTGTACAAA-

AAAGCAGGCTCATGGAAGTTCTGACAAAAATCCTTC-3′ andReverse: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAAT-TGGGCAGTATCTTTCCAGCAAC-3′. The presence of the insertof interest was confirmed by sequencing with the following primers:Forward (T7) 5′-TAATACGACTCACTATAGGG-3′ and Reverse(rBGH) 5′-TAGAAGGCACAGTCGAGG-3′.

Identification of Novel NF1 Missense Mutations

Sequencing of exons plus intronic flanking regions of the NF1gene was performed on lymphocyte DNA samples as previously de-scribed [Thomas et al., 2010, 2012]. Samples were derived fromindividuals with a clinical diagnosis of NF1 based upon stan-dard NIH criteria [Stumpf et al., 1988]. The 15 germline mis-sense mutations in the GRD, identified in a screen of ∼800 pa-tients analyzed in this study, were the only potentially patholog-ical mutations identified in the NF1 gene in these individuals.These mutations were c.3610C>G p.R1204G [Krkljus et al., 1998],c.3610C>T p.R1204W [Ars et al., 2000], c.3827G>A p.R1276Q[Fahsold et al., 2000], c.3902T>G p.L1301R, c.3919A>G p.I1307V,c.3971C>A p.T1324N, c.3980A>G p.E1327G, c.4007A>G p.Q1336R,c.4067A>G p.E1356G [Trovo et al., 2004], c.4171A>G p.R1391G,c.4193T>A p.V1398D, c.4225A>G p.K1409E, c.4235C>G p.P1412R,c.4306A>C p.K1436Q, c.4388C>T p.S1463F (Fig. 1, Table 1). Thetwo positive controls included were c.4173A>T p.R1391S [Upad-hyaya et al., 1997] and c.4267A>G p. K1423E [Li et al., 1992].Nucleotide numbering reflects cDNA numbering with +1 corre-sponding to the A of the ATG initiation codon in the referencesequence NM_000267.3. All variants have been submitted to theLOVD database for NF1 which can be accessed at: http://medgen.ugent.be/LOVD2/home.php?select_db=NF1_germline. Four muta-tions have been previously reported as germline mutations (as in-dicated above) but have not been characterized by functional anal-ysis, whereas the remaining 11 mutations were novel constitutionalchanges. The missense mutation c.4267A>G p.K1423E [Li et al.,1992], which has been previously identified as both a germline anda somatic mutation (in a malignant peripheral nerve sheath tumor[MPNST]) in our laboratory, and p.R1391S [Upadhyaya et al., 1997]were included as independently validated positive controls. The mu-tation p.K1423E was found to be 200–300-fold less active than thewild-type protein [Li et al., 1992], whereas p.R1391S was 300 timesless active (Upadhyaya et al., 1997).

Site-Directed Mutagenesis

The oligonucleotide primers employed for site-directed mutagen-esis were designed individually according to the different mutationsdesired, using the QuikChange primer design program (Agilent,Edinburgh). PCR-based site-directed mutagenesis was performedusing 10 ng/μl plasmid DNA and 125 ng each primer under the fol-lowing typical cycle conditions: 95◦C, 30 sec; Ta (◦C), 1 min; 68◦C,8 min (× 18 cycles). About 1 μl DpnI restriction enzyme (NEB)(10 U/μl) was directly added to each reaction, which was incubatedat 37◦C for 1 hr. Finally, omniMAX bacterial cells (Invitrogen) weretransformed with 2.5 μl DpnI-treated reaction mix and plated ontoLB agar plates containing Ampicillin (50 mg/ml). Plates were in-cubated at 37◦C for 16–24 hr and plasmid DNA was extracted andpurified using the Spin Miniprep Kit (Qiagen, Crawley, West Sus-sex). Each sample was sequenced to confirm the presence of therequired mutation.

About 4.0 μg plasmid DNA containing the mutagenized NF1-GRD was transfected into 6 × 105 HEK293 cells in 6-well platesusing lipofectamine 2000 (Invitrogen) following the manufacturer’s

2 HUMAN MUTATION, Vol. 00, No. 0, 1–10, 2012

Figure 1. Schematic representation of the relative locations, within the GAP-related domain-encoding exons (21–27a), of all 15 missensemutations and two positive controls (∗) analyzed in this study. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A ofthe ATG initiation codon in the reference sequence NM_000267.3.

recommended protocol. Cells were nearing confluency (80%) whentransfected to ensure good transfection efficiency. Cells were serum-starved for 24 hr following transfection and then, prior to lysis, werestimulated for 30 min with EGF [100 ng/mL] (Promokine, UK).Expression of the V5-tagged protein in mutant cell lines, and thosetransfected with a nonmutagenized vector, was assessed by westernblotting using 100 ug cell lysate and NuPAGE Novex 4–12% Bis-Trisgels (Invitrogen). The protein was transferred to Immobilon PVDFmembrane (Millipore) using the Hoefer transfer system, blockedwith 5% (w/v) powdered milk; a V5 mouse monoclonal primaryantibody (1:5,000) (Abcam) was incubated with the membrane at4◦C overnight on an orbital shaker. A rabbit polyclonal secondaryantibody to mouse IgG (HRP) (1:10,000) (Abcam) was incubatedwith the membrane for 30 min at room temperature. The membranewas then developed using an enhanced chemiluminescent (ECL)mix in a 1:1 ratio (GE Healthcare) and exposed to X-ray film.

Ras Enzyme-Linked Immunosorbent Assay

Cells were transfected and stimulated as above. The Ras activa-tion enzyme-linked immunosorbent assay (ELISA) was performedas described in the manufacturer’s instructions (Millipore, cat no:17–497) on 100 μg of the cell lysates that were used for the westernblot analyses. Following addition of the chemiluminescent substrate,the Ras ELISA was evaluated using a microtitre plate luminometer(Applied Biosystems). Relative light units (RLU) were measured foreach sample using the Winglow software (Perkin Elmer) between5 and 60 min after the substrate was added. The ELISA was per-formed three times on each mutant construct using three replicateseach time. Statistical analysis was performed using a t-test with Bon-ferroni correction to allow for multiple testing. After correction formultiple testing, only P < 0.000588 (0.01/17) were considered to besignificant.

Extracellular Signal-Regulated Kinase Western BlotAnalysis

An additional contrasting method was also employed in this studyto determine the level of activation in the Ras-MAPK pathway. Totaland phosphorylated extracellular signal-regulated kinase (ERK) pri-mary antibodies [1:1000] (Cell Signalling, UK) were used to measure

total and phosphorylated ERK in the same cell lysates as were usedfor the ELISA to determine the activity of the Ras-MAPK pathway.Expression was quantified by densitometry using Image J software(http://rsbweb.nih.gov/ij/). Statistical analysis was performed usinga t-test with Bonferroni correction to allow for multiple testing.After correction for multiple testing, only P < 0.001 (0.01/10) wereconsidered to be significant.

Bioinformatic Analysis

Missense variants in the NF1 gene were analyzed (with respectto disruption of protein structure/function) by means of three dif-ferent bioinformatic methods designed to identify disease-causingmissense variants (Table 1). The three methods applied were SIFT[Ng and Henikoff, 2001], MutPred [Li et al., 2009; Mort et al., 2010],and Mutation Taster [Schwarz et al., 2010]. An additional compositevalue, equivalent to the consensus of the three different methods(SIFT, MutPred, and Mutation Taster) was also derived, which wehenceforth refer to as the in silico consensus. For the in silico consensus,the three different lines of evidence (SIFT, MutPred, and MutationTaster) were individually converted to a binary classification (0 or 1),with a “1” representing a deleterious or disease-causing predictionand a “0” representing either a tolerated or “not pathogenic” predic-tion. Giving each line of evidence an equal weighting, the majorityclass (“0” or “1”) for each mutation was used to assign the missensemutation as either pathogenic (majority of “1”s) or not pathogenic(majority of “0”s).

MutPred is designed to model the effect of changes in structuraland/or functional sites between the wild-type and mutant versionsof a given protein. MutPred was, therefore, used to generate hy-potheses as to the underlying molecular mechanism(s) responsiblefor disease pathogenesis associated with the 17 variants (includingthe two positive controls) in this study. The effect of the codingregion variants upon splicing (splice site disruption, cryptic splicesite activation, and exon skipping, as a consequence either of the lossof an exonic splicing enhancer [ESE] and/or the gain of an exonicsplicing silencer [ESS]) was ascertained using Skippy [Woolfe et al.,2010] and a neural network for splice site prediction [Krawczaket al., 2007]. The NI-ESR hexamers [Stadler et al., 2006] (979 ESEsand 496 ESSs) formed the basis of the ESE and ESS motifs used inthis analysis, as this set had previously been identified as providing

HUMAN MUTATION, Vol. 00, No. 0, 1–10, 2012 3

Tabl

e1.

Mis

sens

eVa

rian

tsin

the

NF1

GA

P-Re

late

dD

omai

n,A

sses

sed

with

Bio

info

rmat

icTo

ols

and

byM

eans

ofIn

Vitr

oFu

nctio

nalA

naly

sis

Var

ian

tR

esu

lts

ofR

asE

LISA

assa

y(P

valu

e)R

esu

lts

ofin

silic

oco

nse

nsu

s(S

IFT

,Mut

Pre

d,M

utat

ionT

aste

r)SI

FTpr

edic

tion

(SIF

Tsc

ore)

Mu

tPre

dpr

edic

tion

(pro

babi

lity)

Mu

tPre

dhy

poth

esis

Mu

tati

onta

ster

pred

icti

on(p

roba

bilit

y)P

revi

ousl

yid

enti

fied

/nov

el

c.36

10C

>G

p.R

1204

GPa

thog

enic

∗(2

.15

×10

-14)

Path

ogen

ic(0

,1,1

)T

OLE

RA

TE

D(0

.11)

Path

ogen

ic(0

.84)

Dis

ease

cau

sin

g(0

.99)

[Ars

etal

.,20

00]

c.36

10C

>T

p.R

1204

WPa

thog

enic

∗(4

.49

×10

-09)

Path

ogen

ic(1

,1,1

)D

AM

AG

ING

(0)

Path

ogen

ic(0

.81)

Dis

ease

cau

sin

g(1

.00)

[Krk

ljus

etal

.,19

97]

c.38

27G

>A

p.R

1276

QPa

thog

enic

∗(2

.36

×10

-12)

Path

ogen

ic(1

,1,1

)D

AM

AG

ING

(0)

Path

ogen

ic(0

.98)

Loss

ofca

taly

tic

resi

due

atR

1276

(P=

0.04

04)

Dis

ease

cau

sin

g(1

.00)

[Fah

sold

etal

.,20

00]

c.39

02T

>G

p.L1

301R

Path

ogen

ic(1

.74

×10

-13)

Path

ogen

ic(1

,1,1

)D

AM

AG

ING

(0)

Path

ogen

ic(0

.91)

Dis

ease

cau

sin

g(1

.00)

Nov

elc.

3919

A>

Gp.

I130

7VN

onpa

thog

enic

(9.2

2×

10-0

4)N

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thog

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(0,0

,0)

TO

LER

AT

ED

(0.3

4)Po

lym

orph

ism

(0.4

7)Po

lym

orph

ism

(1.0

0)N

ovel

c.39

71C

>A

p.T

1324

NPa

thog

enic

∗(5

.24

×10

-08)

Not

path

ogen

ic(0

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)T

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RA

TE

D(0

.30)

Poly

mor

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m(0

.36)

Dis

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cau

sin

g(1

.00)

Nov

elc.

3980

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Gp.

E13

27G

Non

path

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ic∗

(2.2

3×

10-0

1)N

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thog

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(0,0

,1)

TO

LER

AT

ED

(0.3

2)Po

lym

orph

ism

(0.4

5)D

isea

seca

usi

ng

(0.9

9)N

ovel

c.40

07A

>G

p.Q

1336

RN

onpa

thog

enic

(6.2

7×

10-0

1)Pa

thog

enic

(0,1

,1)

TO

LER

AT

ED

(0.6

1)Po

ssib

lyPa

thog

enic

(0.6

1)D

isea

seca

usi

ng

(1.0

0)N

ovel

c.40

67A

>G

p.E

1356

GPa

thog

enic

(1.8

9×

10-0

9)Pa

thog

enic

(0,1

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TO

LER

AT

ED

(0.1

6)Po

ssib

lyPa

thog

enic

(0.7

3)D

isea

seca

usi

ng

(1.0

0)[T

rovo

etal

.,20

04]

c.41

71A

>G

p.R

1391

GPa

thog

enic

∗(1

.06

×10

-13)

Path

ogen

ic(1

,1,1

)D

AM

AG

ING

(0)

Path

ogen

ic(0

.92)

Dis

ease

cau

sin

g(1

.00)

Nov

elc.

4173

A>

Tp.

R13

91S

(CO

NT

RO

L)

Path

ogen

ic∗

(6.5

5×

10-1

3)Pa

thog

enic

(1,1

,1)

DA

MA

GIN

G(0

)Pa

thog

enic

(0.9

6)D

isea

seca

usi

ng

(1.0

0)[U

padh

yaya

etal

.,19

97]

c.41

93T

>A

p.V

1398

DPa

thog

enic

∗(3

.39

×10

-10)

Path

ogen

ic(1

,1,1

)D

AM

AG

ING

(0)

Path

ogen

ic(0

.81)

Gai

nof

diso

rder

(P=

0.04

12)

Dis

ease

cau

sin

g(1

.00)

Nov

elc.

4225

A>

Gp.

K14

09E

Non

path

ogen

ic(9

.10

×10

-04)

Not

path

ogen

ic(0

,0,1

)T

OLE

RA

TE

D(0

.98)

Poly

mor

phis

m(0

.44)

Dis

ease

cau

sin

g(1

.00)

Nov

elc.

4235

C>

Gp.

P14

12R

Non

path

ogen

ic(7

.12

×10

-03)

Not

path

ogen

ic(0

,0,1

)T

OLE

RA

TE

D(0

.62)

Poly

mor

phis

m(0

.48)

Dis

ease

cau

sin

g(1

.00)

Nov

elc.

4267

A>

Gp.

K14

23E

(CO

NT

RO

L)

Path

ogen

ic∗

(3.5

3×

10-1

5)Pa

thog

enic

(1,1

,1)

DA

MA

GIN

G(0

)Pa

thog

enic

(0.9

6)Lo

ssof

MoR

Fbi

ndi

ng

(P=

0.00

13)

Dis

ease

cau

sin

g(1

.00)

[Lie

tal

.,19

92]

c.43

06A

>C

p.K

1436

QPa

thog

enic

(1.1

0×

10-0

7)Pa

thog

enic

(1,0

,1)

DA

MA

GIN

G(0

)Po

lym

orph

ism

(0.3

4)D

isea

seca

usi

ng

(0.9

9)N

ovel

c.43

88C

>T

p.S1

463F

Path

ogen

ic(7

.51

×10

-12)

Path

ogen

ic(1

,0,1

)D

AM

AG

ING

(0.0

4)Po

lym

orph

ism

(0.4

6)D

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(1.0

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the strongest signal for identifying exon skipping variants [Woolfeet al., 2010].

The predictions of the bioinformatic methods (SIFT, MutPred,Mutation Taster, and the in silico consensus) were then compared(Supp. Table S1) with the experimentally derived results from thefunctional assays. This allowed evaluation of the efficacy of thesebioinformatic tools specifically in the context of the NF1-GRD. Onelimitation is that this assumes the functional assay to be 100% ac-curate in identifying pathogenic missense variants, which may notbe the case. In total, 15 NF1 missense mutations and two positivecontrols (Table 1) were evaluated by each method using standardbenchmarking scores including the false positive rate (FPR), sensi-tivity, specificity, Matthews correlation coefficient [MCC; Matthews,1975], and accuracy (the mean of sensitivity and specificity scores).

Three-Dimensional Modeling of the NF1 GRD

The three-dimensional (3D) structure of the human NF1-GRD(NP_000258.1; p.1125–1537) was computed using a homologymodeling technique employing the I-TASSER server [Ambrish et al.,2010; Roy et al., 2010]. Several templates were used to derive the pre-dicted 3D structure, including the X-ray crystal structure of the hu-man NF1-GRD (PDB identifier 1nf1A; 2.5 A resolution) comprisingresidues 1,198–1,530 [Scheffzek et al., 1998a,b]. The model used inthis analysis had a C-score of –1.53. The C-score provides a mea-sure of confidence in the quality of a model predicted by I-TASSER.C-scores are typically in the range of –5 to 2, where a higher C-score indicates greater confidence in the accuracy of a given model.The model employed here was annotated using Chimera [Pettersenet al., 2004] with sequence conservation data (Fig. 2A), previouslyidentified (albeit still putative) GAP/Ras interaction sites (Fig. 2B)[Scheffzek et al., 1996] and putative disease-causing mutations char-acterized in this study (Figs. 2C and 3).

Identification of Functionally Important Regions of the GRDUsing Evolutionary Conservation Data

The ConSurf server (http://consurf.tau.ac.il/) was employed toidentify functionally important regions of the human NF1-GRD onthe basis of their evolutionary conservation. This was determinedusing a multiple sequence alignment of the protein sequences of34 unique GRD homologues (including homologues from rhesusmacaque, panda, zebrafish, toad, wasp, Drosophila, and mosquito).The conservation data are displayed in Supp. Table S2 and inFigure 2A.

ResultsIn this study, we have functionally characterized 15 naturally

occurring missense mutations, four of which have been previouslyreported (but not functionally characterized), and two positive con-trol mutations identified in the GRD of human neurofibromin en-coded by the NF1 gene. Taken together, this constitutes the mostcomprehensive functional analysis of the NF1-GRD yet performed.The functional significance of these mutations was assessed by in-troducing them into a vector containing the NF1-GRD and assay-ing activated Ras by means of an ELISA and western blot anal-ysis. This Ras activation assay yielded fluorescence readings, cali-brated in relative light units (RLU), which provided a measure ofthe level of activated Ras associated with the expression of boththe mutant and control constructs. Ten of the 15 analyzed NF1variants (c.3610C>G p.R1204G; c.3610C>T p.R1204W; c.3827G>A

Figure 2. Three-dimensional structural model of the NF1-GRD, an-notated by/with sequence conservation level (A), previously identifiedputative Ras–GRD interaction sites [Scheffzek et al., 1996] (B), and 10potentially disease-causing missense mutations as identified by thefunctional assay used in this study (C).

p.R1276Q; c.3902T>G p.L1301R; c.3971C>A p.T1324N; c.4067A>G,p.E1356G; c.4171A>G p.R1391G; c.4193T>A p.V1398D; c.4306A>Cp.K1436Q, and c.4388C>T p.S1463F [Table 1, Fig. 4]) were deemedto be of functional significance (and hence potentially pathogenic)on the basis that they were associated with significantly elevated(t-test with Bonferroni correction, P < 0.000588) levels of activatedGTP-bound Ras by comparison with the wild-type NF1 protein.Levels of activated GTP-bound Ras were comparable to those asso-ciated with the two positive controls used in this analysis (p.K1423E[Li et al., 1992] and p.R1391S [Upadhyaya et al, 1997]) (Fig. 4).These experimental data indicate that the 10 “pathogenic” GRDmissense variants are associated with a ∼200–300-fold reduction in

HUMAN MUTATION, Vol. 00, No. 0, 1–10, 2012 5

Figure 3. Three-dimensional structural model (showing secondary structure) of the NF1-GRD. The putative RAS–GRD interaction sites aredenoted in purple, whereas the residues affected by the disease-causing missense mutations analyzed in this study are shown in red. Residuescolored black indicate a coincidence of disease-causing mutations (R1276Q, R1391S, R1391G, and K1436Q) with RAS–GRD interaction sites.

Figure 4. Assessment of the functional significance of 15 NF1-GRD missense mutations and two positive control mutations: K1423E and p.R1391Sby an in vitro Ras activation assay. The level of activated GTP-bound Ras associated with the expression of each mutant construct is given inrelative light units (RLU). Statistical analysis of the RLU values generated was performed for each mutant (t-test with Bonferroni correction).Missense mutations associated with significantly elevated (by comparison to wild-type NF1 protein) levels of activated GTP-bound Ras are coloredin red (indicative of significantly elevated RLU values and hence a significantly elevated level of bound GTP in comparison to the wild-type protein)were deemed to be of pathological significance and are designated with a red line. Missense mutations colored in green exhibit a similar levelof activated Ras to that of the wild-type NF1 protein. ∗WT NF1 represents cells transfected with empty vector to demonstrate the basal level ofRas activation in the cells assayed. The p.K1423E and p.R1391S mutations are included as positive controls. Previous analysis has demonstratedthat p.K1423E is associated with a ∼200–300-fold reduction in GRD activity (which would lead to a higher level of intracellular activated Ras) [Liet al., 1992]. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG initiation codon in the reference sequenceNM_000267.3.

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GRD activity (which would be predicted to lead to higher levels ofintracellular activated Ras).

By contrast, five (Fig. 4) of the 15 GRD missense muta-tions studied here were found to be associated with mean flu-orescence readings indicative of GRD activity values comparablewith those associated with wild-type GRD (c.3919A>G p.I1307V;c.3980A>G p.E1327G; c.4007A>G p.Q1336R; c.4225A>G p.K1409E,and c.4235C>G p.P1412R) (P = 0.2) (Fig. 4), suggesting that thesemissense mutations are probably not deleterious to NF1-GRD func-tion. As these five variants were also not predicted in silico to disruptsplicing (see below), we deem them to be putatively nonpathogenic.Expression levels of the V5-tagged NF1 protein as seen by westernblot were found to be the same for all 15 NF1-GRD mutants andtwo control samples indicating that none of the missense mutationshad any measurable impact upon protein levels (although it shouldbe stated that, as the full-length neurofibromin protein was not ex-pressed, we cannot unequivocally determine this in vitro). Hence,we surmised that the differences in expression between mutant pro-teins noted in the Ras ELISA were unlikely to be due simply todifferences in protein level between the cell lysates (Supp. Fig. S1).Phosphorylated and total-ERK expression as determined by west-ern blot analysis of a selection of the 15 variants analyzed (includingthe two positive controls) correlated with the results of the ELISA,demonstrating higher phosphorylated-ERK in those cell lines car-rying NF1 mutants found to be pathogenic by the ELISA method(Supp. Fig. S2). Conversely, the expression of phosphorylated-ERKin cell lines harboring mutations deemed to be nonpathogenic byELISA were similar to those of the cell lines carrying wild-type ERK(Supp. Fig. S2). Total ERK blots confirmed that the expression ofERK was unaffected by these mutations, merely the activation of theRas-MAPK pathway (Supp. Fig. S2).

ERK western blot results were obtainable for 8 of the 15 missensemutations and statistical analysis of these results supported the clas-sification (pathogenic or nonpathogenic) derived from the ELISAresults (Table 1), with the exception of p.K1409E. Therefore, thepathogenic classification was confirmed for p.R1204G, p.R1204W,p.R1276Q, p.T1324N, p.R1391G, and p.V1398D. With regard to thenonpathogenic classification, this was confirmed for the p.E1327Gmissense mutation.

In Silico Prediction of Disease-Causing Missense Variants

For the 10 putatively pathogenic missense mutation (as identi-fied by the functional assay), 90% (nine of the ten) were classifiedcorrectly as pathogenic based on the results of the in silico consensusof SIFT, MutPred, and Mutation Taster (Table 1). Four of the fivemissense variants deemed to be neutral on the basis of the func-tional assay were predicted to be nonpathogenic using the in silicoconsensus (Table 1). Thus, the bioinformatic predictions broadlyconcurred with, and hence supported, the functional assay datawith two exceptions: T1324N was not predicted to be pathogenicby bioinformatic analysis despite the reduction in GRD activity ob-served in the in vitro assay whereas the opposite was the case forQ1336R.

With respect to an effect on splicing (splice site disruption, crypticsplice site activation, and exon skipping via modulation of ESE andESS), two (p.S1463F and p.E1356G) of the 10 putatively pathogenicmissense variants and one of the control mutations (p.K1423E)were predicted to disrupt splicing (Supp. Table S3). The p.S1463Fmutation was predicted using Skippy [Woolfe et al. 2010] to causeexon skipping via the loss of two putative ESE motifs and the creation(gain) of three putative ESS motifs. The loss of two ESE binding sites

combined with the gain of three ESS binding sites consequent to thismutation (p.S1463F), would serve to weaken the “exon definition”and could therefore result in exon skipping. The missense mutationp.E1356G is predicted to activate a cryptic acceptor (3′) splice site,whereas the positive control mutation p.K1423E is predicted toabolish the wild-type donor (5′) splice site. RNA studies, performedrecently in NF1 patients, have, however, suggested that p.K1423Emay not influence NF1 mRNA splicing in vivo (L. Messiaen, June,2012).

Evaluation of In Silico Predicted Disease-CausingPredictions

Using the results of the experimental functional assay, the bioin-formatic measures employed here (SIFT, MutPred, Mutation Taster,and the in silico consensus) were evaluated in the context of the NF1-GRD, using standard benchmarking statistics including the FPR,sensitivity, specificity, MCC (Matthews, 1975) and the accuracy(the mean of the sensitivity and specificity scores). These perfor-mance benchmarking statistics are summarized in Supp. Table S1.The MCC was employed as it represents one of the best availablemeasures of prediction quality; it returns a value between –1 and +1,with a coefficient of –1 representing the worst possible prediction, 0a random prediction, and +1 a perfect prediction. Using the MCC toevaluate the three different bioinformatic measures employed here(SIFT, MutPred, Mutation Taster) and the in silico consensus of allthree tools combined, we found that all methods worked well, withMCC values in the range 0.39–0.72. However, the in silico consen-sus (MCC = 0.72), a composite value derived from the individualpredictions of SIFT, MutPred, and Mutation Taster, outperformedeach method on its own. As far as the individual methods (SIFT,MutPred, and Mutation Taster) were concerned, we found SIFT tobe the most effective tool with an MCC of 0.68 (Supp. Table S1).It should, however, be noted that this evaluation was based upon arelatively small sample of functionally characterized variants fromthe NF1 gene and therefore any extrapolation to larger collectionsof missense mutations derived from other genes would be inappro-priate. For an overall performance evaluation of SIFT, MutPred, andMutation Taster, the reader is referred to the original publicationsor recent reviews [see, e.g., Thusberg et al., 2011].

Analysis of NF1-GRD Evolutionary Conservation

On the basis of a multiple sequence alignment of 34 unique GAP-related domain homologues, we ascertained that 55% (227/413) ofthe NF1-GRD residues are evolutionarily conserved (conservationscore ≥6) (Supp. Table S2). Of these conserved residues, 48 residuesin the NF1-GRD exhibited very high evolutionary conservation(conservation score = 9) and are likely to be “key” residues criticalto the structure and function of the NF1-GRD (Supp. Table S2,Fig. 2A). Indeed, both the positive control mutations (p.R1391S andp.K1423E) reside in the highly conserved “key” residues identifiedhere. Of the remaining 15 missense variants analyzed in this study,three (p.R1276Q, p.R1391G, and p.K1436Q) were also located inhighly conserved “key” residues. All three of these “key” residuevariants were also predicted to be pathogenic with the functionalassay. Therefore, no nonpathogenic (as determined by the functionalassay) variants were found to be located in highly conserved “key”residues. With respect to the 17 residues previously identified asbeing putative Ras–GRD interaction sites [Scheffzek et al., 1996],over half (53%; 9 out of 17 residues) of these are located at highlyconserved “key” residues.

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Computational Structural Analysis of NF1 GRD Variants

To investigate the underlying mechanism of disease pathogene-sis, a 3D structural model of the NF1-GRD and a prediction tool,MutPred, were applied. MutPred is designed to model the effect ofchanges in structural and/or functional sites between the wild-typeand mutant versions of a given protein. Three of the putative disease-causing mutations (R1276Q, R1391G, and K1436Q) plus one pos-itive control (p.R1391S) are located directly at the NF1-GRD–Rasinterface (Fig. 2B and C). Because these four mutations are locateddirectly at the protein–protein interaction interface, it is highly likelythat they represent loss-of-function mutations, which either inter-fere with or abolish the transient protein–protein interactions be-tween the NF1-GRD and Ras. The MutPred in silico hypothesis forthe R1276Q mutation (Table 1) provides additional support for thispostulate since it predicts the loss of a catalytic residue at R1276(P = 0.04). Visualization of the 3D structural model of the NF1-GRD (Fig. 2B and C) further reveals that, with the exception of twodisease-causing mutations located in the same amino acid residue(R1204G and R1204W), all the putative disease-causing mutationsidentified in this study (by means of the functional assay), residewithin or in close proximity to the putative Ras interaction/bindingsites. On the basis of the 3D structure, residue R1204 is buried(solvent inaccessible) implying that both R1204G and R1204W rep-resent mutations, which could abolish/reduce Ras binding indirectlyvia disruption of protein stability.

DiscussionAlthough 23.7% of all known NF1 mutations are of the missense

type (HGMD; http://www.hgmd.org), missense mutations remainone of the least well characterized types of mutational lesion re-ported from the NF1 gene. This is in part due to the paucity ofrecognizable domains with specified functions within the neurofi-bromin molecule, and in part because of the lack of biological as-says for neurofibromin activity. In this study, we set out to assessthe pathogenicity of a series of 15 inherited missense mutationsidentified in the GAP-related domain (GRD), the major domainwithin the neurofibromin protein. Eleven of these 15 mutationsare novel, whereas 4 have been previously reported although notinvestigated functionally: c.3610C>G p.R1204G [Ars et al., 2000],c.3610C>T p.R1204W [Krkljus et al., 1998], c.3827G>A p.R1276Q[Fahsold et al., 2000], c.3902T>G p.L1301R, c.3919A>G p.I1307V,c.3971C>A p.T1324N, c.3980A>G p.E1327G, c.4007A>G p.Q1336R,c.4067A>G p.E1356G [Trovo et al., 2004], c.4171A>G p.R1391G,c.4193T>A p.V1398D, c.4225A>G p.K1409E, c.4235C>G p.P1412R,c.4306A>C p.K1436Q, and c.4388C>T p.S1463F. Two of these inher-ited missense mutations have, however, been previously identifiedas somatic mutations in neurofibromas (c.3827G>A p.R1276Q andc.4388C>T; p.S1463F) ([Fahsold et al., 2000; Thomas et al., 2012];Upadhyaya, unpublished result).

The rationale for performing functional analysis on these mis-sense mutations was to obtain new insights into the structure andfunction of the NF1-GRD and the mechanism by which it binds Rasthereby attenuating Ras activity. Our results both concur with, andextend, previous findings. There are a number of different mech-anisms which could be responsible for the disruption of Ras–Ras–GAP interactions in the context of a neurofibromin molecule com-promised by virtue of it harboring missense mutations within itsGRD. NF1-GRD missense variants may either be completely un-able to bind Ras, binding may be impaired, or binding to RasGTPcould be normal but the ability of the mutant neurofibromin to

stimulate Ras may be reduced. The cellular level of mutant neurofi-bromin could also be greatly reduced if the GRD missense muta-tions rendered the mutant neurofibromin molecule unstable (whichwould lead to reduced Ras binding and consequently a higher levelof intracellular activated Ras). Our work identifies 48 highly con-served “key” residues (Fig. 2A), representing ∼12% of the aminoacid residues within the NF1-GRD. These “key” residues are eitherburied within the protein or are exposed on the surface of the pro-tein. We find an abundance of these “key” residues to be locatedat, or in the close vicinity of, Ras–GRD interaction sites (Fig. 2B).Furthermore, all of the putative disease-causing mutations identi-fied here (except p.R1204G and p.R1204W) are located at (R1276Q,R1391S, R1391G, and K1436Q) or in the close vicinity of (p.L1301R,p.T1324N, p.E1356G, p.V1398D, p.K1423E, and p.S1463F) the Ras–GRD interaction sites. Those mutations, which reside on the proteinsurface in the vicinity of the NF1-GRD, are more likely to directlyimpair or abolish the binding of this domain with Ras. By contrast,mutations embedded deep within the protein structure (p.R1204Gand p.R1204W) may impact upon protein structure stability andabolish Ras–GRD binding indirectly.

Because disruption to splicing is a common mechanism respon-sible for inherited disease [Sterne-Weiler et al., 2011], missensevariants should always be assessed for any impact on the splicingphenotype alongside any assessment of protein structure/functiondisruption. For three of the probable disease-causing mutationsanalyzed here (p.E1356G, p.K1423E, and p.S1463F), we also iden-tified a potential influence on the mRNA phenotype; thus, thesemis-spliced transcripts may be responsible solely for disruption ofprotein function or in combination with the correctly spliced tran-scripts containing the harmful substitution (e.g., 50% of transcriptsmis-spliced, with the other 50% of transcripts spliced correctly butcontaining the missense mutation shown to disrupt Ras–GAP bind-ing). Taken together, the combination of functional, bioinformaticand structural analyses allowed us to dissect the molecular basis ofdysregulated NF1-GAP function.

The biological functions of different regions of the NF1-GRDhave already been investigated: the Thr finger loop and the FLRare associated with catalytic and binding domains, whereas theso-called variable loop region accounts for the specificity of theRas–GAP interaction [Ahmadian et al., 1996]. To date, a total of150 disease-causing mutations have been reported in the NF1-GRD(NP_000258.1; p.1125–1537) of which 25 are missense; prior to thisstudy, only four of these missense mutations had been functionallycharacterized [Ahmadian et al., 2003; Klose et al., 1998; Li et al.,1992; Morcos et al., 1996; Poullet et al., 1994; Upadhyaya et al.,1997]. Further, the analyses of the pathogenicity of these NF1-GRDmissense mutations so far performed have been somewhat limitedin their scope. Hence, this current study represents the largest anal-ysis to date. The PCR- and ELISA-based functional assay deployedhere also represents a substantial improvement on previous anal-yses because it is simple, rapid, and cost-effective, and it does notrequire either radiolabeled nucleotides [Li et al., 1992; Upadhyayaet al., 1997] or the construction of extensive libraries of vector con-structs [Morcos et al., 1996]. Further, ELISA offers the potential ofa quantitative assay, and hence represents a more reliable method todetermine protein function.

As evolutionary conservation of the neurofibromin sequence isnot confined to the GRD, it is possible that amino acid residueslocated adjacent to the GRD sensu stricto may also serve to mod-ulate the GAP activity of neurofibromin. Indeed, there is someevidence that the cysteine/serine-rich domain (CSRD), located atthe N-terminal of neurofibromin, may be able to modulate GAPactivity following serine phosphorylation [Mangoura et al., 2006].

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Further, the pre-GRD region of neurofibromin is known to alterthe expression of genes involved in cell adhesion and migration,and acts as a negative regulator of the Rac1/Pak1/LIMK1/cofilinpathway [Starinsky-Elbaz et al., 2009]. This may provide an expla-nation for why Q1336R is predicted (by bioinformatic tools) to bepathogenic but does not appear from in vitro functional analysisto impact adversely on GRD function (and vice versa for T1324N).It should, however, be appreciated that, in this functional assay, weare not dealing with full-length neurofibromin in vivo but rather ashort GRD construct in vitro; it may, therefore, be that the CSRDcould determine whether or not a given GRD mutation influencesGRD function in vivo. In addition to identifying the mechanismsunderlying the aberrant function of a mutant protein, the concur-rent use of bioinformatic analysis also allows us to comment on invitro activities of mutant cell lines

Although germline mutations involving K1423 have been re-ported multiple times in the literature [Li et al., 1992; Morcos et al.,1996; Poullet et al., 1994], these lesions have also been noted inthe soma, having previously been identified in both NF1-associatedneurofibromas and in an MPNST in our laboratory (Upadhyayaet al., unpublished result). Li et al. (1992) found that the K1423Emutation resulted in a 200–300-fold reduction in GAP activity. It isthought that the K1423 residue forms an intramolecular salt bridgewith E1437 in the α7/variable loop, thereby establishing a key com-ponent of the Ras-Ras–GAP interaction. Disruption of this saltbridge by mutation of the K1423 residue would introduce negativecharges, which could interfere with the Ras–NF1-GRD interaction[Klose et al., 1998]. Because K1423 is located at some distance fromthe Ras active site, it is unlikely to be directly involved in catalysis butcould be involved in determining the specificity of neurofibrominfor Ras-GTP [Ahmadian et al., 2003]. In our own study, the K1423Emutation was used as a positive control; we confirmed a similar re-duction in GAP activity to that originally reported by Li et al. (1992),thereby strengthening the available evidence for the functional sig-nificance of this residue, at the same time as validating our in vitroassay. The results of our bioinformatic analysis broadly concurredwith those of the functional and structural analyses. Further, Mut-Pred not only predicted that p.K1423E is disease causing (P = 0.96),it also generated a confident in silico hypothesis that the underly-ing mechanism of pathogenesis was related to the loss of molecularrecognition features (MoRFs; P = 0.0013). MoRFs are intrinsicallydisordered regions that possess molecular recognition and bindingfunctions [Vacic et al., 2007]. We surmise that the loss of MoRFs asa consequence of the p.K1423E mutation would be likely to impactupon the recognition and/or binding between Ras and the GRD.

The R1391 residue is known to be a supporting arginine residuewithin the FLR region, responsible for the orientation and stabiliza-tion of the finger loop without directly contacting the active site inRas [Ahmadian et al., 2003; Scheffzek et al., 1996, 1997]. The “fingerloop” lines the active site of Ras, exposing a highly conserved (Supp.Table S2) arginine residue (p.R1276); this residue was previouslyreported to be a NF1-GRD–Ras interaction site [Scheffzek et al.,1996]. The R1391 residue has been well studied in several differ-ent mutational contexts, including R1391K [Skinner et al., 1991]and R1391S [Upadhyaya et al., 1997]. The latter study measuredthe amount of radioactivity that remained bound to H-Ras, whichwas proportional to the extent of NF1-GRD-mediated GTP hydrol-ysis. The GRD harboring the R1391S missense mutant was foundto be ∼300-fold less active than the wild-type NF1-GRD [Upad-hyaya et al., 1997], a finding which we confirmed here using ourown assay which is novel in the context of NF1 missense mutationanalysis (Fig. 4, Table 1). Further, the third variant at this residue(R1391G) was found to exhibit the highest level of GTP-bound Ras

(6 × 106 RLU, corresponding to a ∼400-fold reduction in GAP ac-tivity) of all the 15 missense mutations analyzed in this study. Thepathogenicity of the R1391G variant was also predicted by all threebioinformatic methods employed (SIFT, MutPred, and MutationTaster). The p.R1391 residue is a putative NF1-GRD–Ras interac-tion site [Scheffzek et al., 1996] that is both highly conserved (Supp.Table S2) and known to be of fundamental significance to NF1-GAPfunction (Ahmadian et al., 1997; Sermon et al., 1998).

Both structural and biochemical studies have provided evidenceto support a key role for the arginine finger in NF1-GRD functionthrough its insertion into the active site of Ras. This process is knownto stabilize the transition state for the GTPase reaction [Ahmadianet al., 2003; Resat et al., 2001]. p.R1276, one of the most importantresidues to be analyzed in this study, is highly conserved (Supp.Table S2) and serves as the arginine finger which contacts the Ras-GTP/GDP-binding site to stabilize the transition state of the GTPasereaction. The R1276Q mutation studied here has been identifiedboth as a germline and a somatic mutation [Thomas et al., 2012]. Inaddition, an R1276P substitution has been identified in a malignantschwannoma [Klose et al., 1998]. Previous studies have determinedthat although the R1276P substitution only results in a slight reduc-tion in binding affinity for Ras (6.6-fold), it severely compromisesGTP hydrolysis (by some 8,000-fold) [Klose et al., 1998]. We foundthat R1276Q gives rise to an increase in GTP-bound Ras comparedto that observed with wild-type NF1-GRD. However, using our as-say, we cannot distinguish between an effect on Ras-binding affinityand an effect on GTP hydrolysis. This notwithstanding, p.R1276constitutes a highly conserved (Supp. Table S2) putative GRD–Rasinteraction site, which MutPred predicted to be of crucial impor-tance to the catalytic activity of neurofibromin. It should, however,be noted that a near normal GAP activity level associated with agiven NF1-GRD variant may not necessarily provide evidence for aneutral change with no pathological significance.

A combination of functional, bioinformatic and structural anal-yses, therefore, extends previous work in identifying the sites ofNF1-GRD–Ras interaction and improving their definition (p.1236,p.1272, p.1276, p.1277, p.1278, p.1279, p.1286, p.1382, p.1390,p.1391, p.1394. p.1395, p.1423, p.1430, p.1432, p.1436, p.1437).Indeed, four of the disease-causing mutations (R1276Q, R1391S,R1391G, and K1436Q) analyzed here occur at putative NF1-GRD–Ras interaction sites. This study, however, also indicates that thereare several key residues, within the GRD but outside of these pu-tative NF1-GRD–Ras interaction sites, which are crucial for GRDfunction. Relatively few NF1-GRD substitutions were found to haveeither no impact or only an intermediate effect on GAP activity.

The activity of the GRD is clearly key to the role of neurofibrominin the regulation of the Ras-MAPK pathway. Finally, although GRDmissense mutations represent only ∼15% of the total number ofgermline NF1 microlesions, it is worth exploring the potential fortherapeutic approaches specifically tailored to those patients whospecifically harbor GRD missense mutations. The introduction ofsmall molecules able to mimic GAP activity [Klose et al., 1998] intocells derived from the neural crest could in principle be used torestore neurofibromin function (or at least NF1-GAP function) insuch patients.

Acknowledgments

We thank Moira Macdonald for providing information on sequence variants,which were identified from genetic screens of NF1 patients in the CardiffNHS Diagnostic Laboratory. We would also like to thank Kayleigh Dodd forher expert technical assistance.

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